SUMMARY OF WORK This research area involves the study of embryonic stem and induced pluripotent stem cells prior to and during differentiation to cardiomyocytes. For these studies, we employ embryonic stem (ES) cells (R1, D3, HES2), embryonic germ cells (EG-1) embryonic carcinoma cells (P19), and several lines of induced pluripotent stem cells from mouse. Past accomplishments include establishment of efficient in vitro systems to generation of cardiomyocytes from ES cells in vitro, and the analysis of ryanodine type 2 deficient ES cells and their effects on cardiomyocytes. Selection protocols (most recently with a cardiac-restricted portion of the Na/Ca exchanger promoter) have also permitted the isolation of purified cardiomyocytes from these heterogeneous cultures. Recently, we established an in vitro model consisting of monolayer cultures of highly proliferative embryonic stem (ES) cell-derived CMs that can be employed that facilitate the analysis of cell cycle control mechanisms. Separately, the research is aimed at generating cardiac-lineage specific cells to understand the role of regulatory proteins in the formation of cardiomyocytes in vitro. This includes the analysis of BMP/SMAD signalling and the inductive roles of ascorbate and suramen. The effects of suramen on pacemaker-like cell induction are in press (Weise et al, Int J Cardiol. 2009 Sep 21. Epub ahead of print), and the data on BMP signaling and ascorbate are being prepared for publication. An additional focus of this basic research effort is devoted to the improved viability of these cells during in vitro cultivation conditions. For this we have projects evaluating the effects of Matrigel on cardiomyocyte differentiation and stability, which has led to a focus on the roles of p53 and Mdm2. More recently, we have collaborated with groups at UC Davis/Mount Sinai, NY to examine microRNA expression during development and differentiation. We have begun isolating (and targeting) cells to select sub-populations of cardiac progenitor cells based on cell surface markers that may be more appropriate for cellular based therapies. A proof of principal study in C2 myoblasts was recently published that illustrates the proteomic based methodology employed in these studies, which have now expanded to examine undifferentiated ES cells. By studying the basic biology of embryonic stem cells and identifying methods for isolating sub-populations, we hope to delineate novel mechanisms responsible for cardiomyocyte development and renewal, and apply these results to improve cellular based therapies that may be applicable to man.